Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised patients and results in an annual death toll of over 600,000. Cryptococcosis is contracted by inhalation of the infectious particle, which leads to a primary pulmonary infection. C. neoformans is a facultative intracellular pathogen. After initial engulfment by phagocytic cells in the lungs it may lyse or exit these cells and disseminate throughout the mammalian host, either in the bloodstream or by entering additional phagocytes. The consequence of dissemination to the brain is lethal meningitis. Several C. neoformans virulence factors have been implicated in the pathogenesis of this disease, including formation of the protective pigment melanin, the presence on fungal cells of an extensive polysaccharide capsule, and secretion of certain proteins. Host factors also play a role in infection, and host phagocytes have been implicated in pathogen latency and dissemination. Despite the demonstration of host phagocytic cell involvement in this disease, there has been no effort to systematically identify and study genes that are significant for the infection process. In preliminary studies, I developed and optimized a high-throughput imaging assay to efficiently evaluate the interactions between C. neoformans and RNAi-treated human macrophage-like cell lines. I then screened a subset of host genes to identify factors important in these interactions, by using commercially available siRNA libraries to down-regulate expression of genes encoding 901 human kinases and phosphatases in the host cells and then performing interaction assays. This screen identified 8 genes as having specific roles in host:fungal interactions; in this application I propose to study two of them in detail. The broad, long-term objectives of this project are to identify host genes that play a significant role in fungal pathogenesis and to discover their mechanisms of involvement. Aim 1 is designed to test the roles of two candidate host genes in models relevant to pathogenesis, including mouse primary cells in vitro and models of infection. Aim 2 is designed to elucidate the cellular mechanisms by which the products of the two host genes of interest influence host:fungal interactions. These studies will elucidate key aspects of pathogenesis of this important disease by defining how specific host factors affect the interaction of phagocytic cells with C. neoformans. This work will increase understanding of cryptococcosis and potentially aid in the development of therapeutic or preventive strategies to fight this disease.